Renal tubular epithelial injury results in dramatic cellular phenotypic changes including alternations in morphology, cytoskeletal organization, and induction of gene expression thought to be critical for regulating the cell's early response to injury. Significant progress has been made in defining the components and understanding the function of intracellular signal transduction pathways that lead to regulation of similar processes in other systems. The stress activated protein kinase pathway and in particular SAPKs (stress activated protein kinases/c-jun N-terminal kinases) are rapidly activated by multiple cell stressing stimuli including ischemia/reperfusion induced injury of the kidney. Moreover, the SAPKs are activated by GTP-bound Rac1 and Cdc42Hs, Rho- like GTPases that were originally described as regulators of cytoskeletal organization. Therefore, it has been proposed that regulation of signal transduction pathways leading to SAPKs activation may initiate part of the kidney's early response to injury. We have identified, cloned from embryonic kidney, and initially characterized a novel serine/threonine protein kinase called DLK. DLK is a member of a structurally unique subfamily of protein kinases named mixed lineage kinases. As presented herein, DLK potently activates p46SAPK and p38 mapk but not ERK2 when overexpressed in cell culture. DLK appears to participate as a proximal activator of the SAPK pathway and may be a proximal effector for Rac1 and Cdc42Hs. In normal adult kidney, DLK is expressed in the proximal tubular epithelium where it is present only within a subapical subcellular compartment. Given its localization within the kidney, its ability to activate SAPK and p38mapk, and its potential role as an effector of Rac1 and Cdc42Hs, we hypothesize that DLK represents a proximal component of a signal transduction pathway or pathways that participates in modulating the response of the tubular epithelium to cellular stress or injury. This proposal seeks primarily to investigate the fundamental biochemistry and regulation of DLK and will begin to apply these findings to the proximal tubular epithelium. Specifically, this project will: Specific Aim 1: Identify the specific downstream substrate(s) of DLK and define the downstream pathway through which DLK activates p46SAPK and p38 mapk. Specific Aim 2: Investigate the regulation of DLK activity both by potential upstream stimuli and by protein phosphatase 2B (calcineurin). Specific Aim 3: Investigate the properties of DLK's leucine zipper domain and identify leucine zipper domain interacting proteins.